Method for selecting therapeutic agents for cancer

ABSTRACT

The present invention discloses in vitro methods for screening candidate therapeutic and/or chemotherapeutic agents for in vivo efficacy in a specific patient. The invention also includes methods for determining the optimal dosage of a therapeutic agent for a specific patient. The methods include the use of cohesive multicellular particulates of tissue, rather than enzymatically dissociated cell suspensions or preparations, to prepare tissue culture monolayers representative of in vivo cell populations.

This is a divisional application of U.S. application Ser. No. 10/205,887(filed Jul. 26, 2002), now U.S. Pat. No. 6,887,680 (issued May 3, 2005),which is a continuation of U.S. application Ser. No. 09/040,161 (filedMar. 17, 1998), now U.S. Pat. No. 6,900,027 (issued May 31, 2005), whichis a continuation of U.S. application Ser. No. 08/679,056 (filed Jul.12, 1996), now U.S. Pat. No. 5,728,541 (issued Mar. 17, 1998), all ofwhich are hereby incorporated herein by reference in their entirety.

FIELD OF THE INVENTION

The invention relates to screening and testing of active agents,including chemotherapeutic agents, to predict potential efficacy inindividual patients in whom treatment with such agents is indicated.

INTRODUCTION

All active agents including chemotherapeutic active agents are subjectedto rigorous testing as to efficacy and safety prior to approval formedical use in the United States. Methods of assessing efficacy haveincluded elaborate investigations of large populations in double blindstudies as to a given treatment method and/or active agent, withconcommitant statistical interpretation of the resulting data, but theseconclusions are inevitably generalized as to patient populations takenas a whole. In many pharmaceutical disciplines and particularly in thearea of chemotherapy, however, the results of individual patient therapymay not comport with generalized data—to the detriment of the individualpatient. The need has been long recognized for a method of assessing thetherapeutic potential of active agents, including but not limited tochemotherapeutic agents, for their efficacy as to a given individualpatient, prior to the treatment of that patient.

Prior art assays already exist which expose malignant tissue of varioustypes to a plurality of active agents, for the purpose of assessing thebest choice for therapeutic administration. For example, in Kruczynski,A., et al., “Evidence of a direct relationship between the increase inthe in vitro passage number of human non-small-cell-lung cancerprimocultures and their chemosensitivity,” Anticancer Research, vol. 13,no. 2, pp. 507-513 (1993), chemosensitivity of non-small-cell-lungcancers was investigated in in vivo grafts, in in vitro primoculturesand in commercially available long-term cancer cell lines. The increasein chemosensitivity was documented and correlated with morphologicalchanges in the cells in question. Sometimes animal model malignant cellsand/or established cell cultures are tested with prospective therapyagents, see for example Arnold, J. T., “Evaluation of chemopreventiveagents in different mechanistic classes using a rat tracheal epithelialcell culture transformation assay,” Cancer Res., vol. 55, no. 3, pp.537-543 (1995).

When actual patient cells are used to form in vitro assays focussed onindividual patients, in typical prior art processes the cells areharvested (biopsied) and trypsinized (connective tissue digested withthe enzyme trypsin) to yield a cell suspension suitable for conversionto the desired tissue culture form. The in vitro tissue culture cellcollections which result from these techniques are generally plagued bytheir inability accurately to imitate the chemosensitivity of theoriginal tumor or other cell biopsy. Standard cloning and tissue culturetechniques are moreover excessively complicated and expensive for use ina patient-by-patient assay setting. A need thus remains for a techniqueof tissue culture preparation which provides cell cultures, for drugscreening purposes, in which after simple preparation the cell culturesreact in a manner equivalent to their in vivo reactivity, to enable drugor chemotherapeutic agent screening as to a particular patient for whomsuch screening is indicated.

SUMMARY OF THE INVENTION

In order to meet this need, the present invention is an improved systemfor screening a multiple of candidate therapeutic or chemotherapeuticagents for efficacy as to a specific patient, in which a tissue samplefrom the patient is harvested, cultured and separately exposed to aplurality of treatments and/or therapeutic agents for the purpose ofobjectively identifying the best treatment for the cultured cellsobtained from the patient. Specific method innovations such as tissuesample preparation techniques render this method practically as well astheoretically useful. One particularly important tissue samplepreparation technique is the initial preparation of cohesivemulticellular particulates of the tissue sample, rather thanenzymatically dissociated cell suspensions or preparations, for initialtissue culture monolayer preparation. With respect to the culturing ofmalignant cells, for example, it is believed (without any intention ofbeing bound by the theory) that by maintaining the malignant cellswithin a multicellular particulate of the originating tissue, growth ofthe malignant cells themselves is facilitated versus the overgrowth offibroblasts or other cells which tends to occur when suspended tumorcells are grown in culture. Practical monolayers of cells may thus beformed to enable meaningful screening of a plurality of treatmentsand/or agents. Growth of cells is monitored to ascertain the time toinitiate the assay and to determine the growth rate of the culturedcells; sequence and timing of drug addition is also monitored andoptimized. By subjecting uniform samples of cells to a wide variety ofactive agents (and concentrations thereof), the most efficacious agentcan be determined. For assays concerning cancer treatment, a two-stageevaluation is contemplated in which both acute cytotoxic and longer terminhibitory effect of a given anti-cancer agent are investigated.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a system for screening a multiple of candidatetherapeutic or chemotherapeutic agents for efficacy as to a specificpatient, in which a tissue sample from the patient is harvested andseparately exposed to a plurality of treatments and/or therapeuticagents for the purpose of objectively identifying the best treatment oragent. Specific method innovations such as tissue sample preparationtechniques render this method practically as well as theoreticallyuseful. One particularly important tissue sample preparation techniqueis the initial preparation of cohesive multicellular particulates(explants) of the tissue sample, rather than enzymatically dissociatedcell suspensions or preparations, for initial tissue culture monolayerpreparation. Cell growth, and sequence and timing of drug addition, aremonitored and optimized.

An important application of the present invention is the screening ofchemotherapeutic agents and other antineoplastic therapies againsttissue culture preparations of malignant cells from the patients fromwhom malignant samples are biopsied. Related anti-cancer therapies whichcan be screened using the inventive system are both radiation therapyand agents which enhance the cytotoxicity of radiation, as well asimmunotherapeutic anti-cancer agents. Screening processes for treatmentsor therapeutic agents for nonmalignant syndromes are also embracedwithin this invention, however, and include without limitation agentswhich combat hyperproliferative syndromes, such as psoriasis, or woundhealing agents. Nor is the present efficacy assay limited only to thescreening of active agents which speed up (healing) or slow down(anti-cancer, anti-hyperproliferative) cell growth because agentsintended to enhance or to subdue intracellular biochemical functions maybe tested in the present tissue culture system also. For example, theformation or blocking of enzymes, neurotransmitters and otherbiochemicals may be screened with the present assay methods prior totreatment of the patient.

When the patient is to be treated for the presence of tumor, in thepreferred embodiment of the present invention a tumor biopsy of >100 mgof non-necrotic, non-contaminated tissue is harvested from the patientby any suitable biopsy or surgical procedure known in the art. Biopsysample preparation generally proceeds as follows under a Laminar FlowHood which should be turned on at least 20 minutes before use. Reagentgrade ethanol is used to wipe down the surface of the hood prior tobeginning the sample preparation. The tumor is then removed, understerile conditions, from the shipping container and is minced withsterile scissors. If the specimen arrives already minced, the individualtumor pieces should be divided into four groups. Using sterile forceps,each undivided tissue quarter is then placed in 3 ml sterile growthmedium (Standard F-10 medium containing 17% calf serum and a standardamount of Penicillin and Streptomycin) and systematically minced byusing two sterile scalpels in a scissor-like motion, or mechanicallyequivalent manual or automated opposing incisor blades. Thiscross-cutting motion is important because the technique creates smoothcut edges on the resulting tumor multicellular particulates. Preferablybut not necessarily, the tumor particulates each measure 1 mm³. Aftereach tumor quarter has been minced, the particles are plated in cultureflasks using sterile pasteur pipettes (9 explants per T-25 or 20particulates per T-75 flask). Each flask is then labelled with thepatient's code, the date of explantation and any other distinguishingdata. The explants should be evenly distributed across the bottomsurface of the flask, with initial inverted incubation in a 37° C.incubator for 5-10 minutes, followed by addition of about 5-10 mlsterile growth medium and further incubation in the normal, non-invertedposition. Flasks are placed in a 35° C., non-CO₂ incubator. Flasksshould be checked daily for growth and contamination. Over a period of afew weeks, with weekly removal and replacement of 5 ml of growth medium,the explants will foster growth of cells into a monolayer. With respectto the culturing of malignant cells, it is believed (without anyintention of being bound by the theory) that by maintaining themalignant cells within a multicellular particulate of the originatingtissue, growth of the malignant cells themselves is facilitated versusthe overgrowth of fibroblasts (or other unwanted cells) which tends tooccur when suspended tumor cells are grown in culture.

The use of the above procedure to form a cell monolayer culturemaximizes the growth of malignant cells from the tissue sample, and thusoptimizes ensuing tissue culture assay of chemotherapeutic action ofvarious agents to be tested. Enhanced growth of actual malignant cellsis only one aspect of the present invention, however; another importantfeature is the growth rate monitoring system used to oversee growth ofthe monolayer once formed. Once a primary culture and its derivedsecondary monolayer tissue culture has been initiated, the growth of thecells is monitored to ascertain the time to initiate the chemotherapyassay and to determine the growth rate of the cultured cells.

Monitoring of the growth of cells is conducted by counting the cells inthe monolayer on a periodic basis, without killing or staining the cellsand without removing any cells from the culture flask. The counting maybe done visually or by automated methods, either with or without the useof estimating techniques known in the art (counting in a representativearea of a grid multiplied by number of grid areas, for example). Datafrom periodic counting is then used to determine growth rates which mayor may not be considered parallel to growth rates of the same cells invivo in the patient. If growth rate cycles can be documented, forexample, then dosing of certain active agents can be customized for thepatient. The same growth rate can be used to evaluate radiationtreatment periodicity, as well. It should be noted that with the growthrate determinations conducted while the monolayers grow in their flasks,the present method requires no hemocytometry, flow cytometry or use ofmicroscope slides and staining, with all their concommitant labor andcost.

Protocols for monolayer growth rate generally use a phase-contrastinverted microscope to examine culture flasks incubated in a 37° C. (5%CO₂) incubator. When the flask is placed under the phase-contrastinverted microscope, ten fields (areas on a grid inherent to the flask)are examined using the 10× objective, with the proviso that the tenfields should be non-contiguous, or significantly removed from oneanother, so that the ten fields are a representative sampling of thewhole flask. Percentage cell occupancy for each field examined is noted,and averaging of these percentages then provides an estimate of overallpercent confluency in the cell culture. When patient samples have beendivided between two or among three or more flasks, an average cell countfor the total patient sample should be calculated. The calculatedaverage percent confluency should be entered into a process log toenable compilation of data—and plotting of growth curves—over time.Monolayer cultures may be photographed to document cell morphology andculture growth patterns. The applicable formula is:

${{Percent}\mspace{14mu}{confluency}} = \frac{{estimate}\mspace{14mu}{of}\mspace{14mu}{the}\mspace{14mu}{area}\mspace{14mu}{occupied}\mspace{14mu}{by}\mspace{14mu}{cells}}{{total}\mspace{14mu}{area}\mspace{14mu}{in}\mspace{14mu}{an}\mspace{14mu}{observed}\mspace{14mu}{field}}$As an example, therefore, if the estimate of area occupied by the cellsis 30% and the total area of the field is 100%, percent confluency is30/100, or 30.

Adaptation of the above protocol for non-tumor cells is straightforwardand generally constitutes an equivalent procedure.

Active agent screening using the cultured cells does not proceed in theinitial incubation flask, but generally proceeds using plates such asmicrotiter plates. The performance of the chemosensitivity assay usedfor screening purposes depends on the ability to deliver a reproduciblecell number to each row in a plate and/or a series of plates, as well asthe ability to achieve an even distribution of cells throughout a givenwell. The following procedure assures that cells are reproduciblytransferred from flask to microtiter plates, and cells are evenlydistributed across the surface of each well.

The first step in preparing the microtiter plates is, of course,preparing and monitoring the monolayer as described above. The followingprotocol is exemplary and susceptible of variation as will be apparentto one skilled in the art. Cells are removed from the culture flask anda cell pellet is prepared by centrifugation. The cell pellet derivedfrom the monolayer is then suspended in 5 ml of the growth medium andmixed in a conical tube with a vortex for 6 to 10 seconds. The tube isthen rocked back and forth 10 times. A 36 μl droplet from the center ofthe conical tube is pipetted onto one well of a 96 well plate. A freshpipette is then used to pipette a 36 μl aliquot of trypan blue solution,which is added to the same well, and the two droplets are mixed withrepeated pipette aspiration. The resulting admixture is then dividedbetween two hemocytometer chambers for examination using a standardlight microscope. Cells are counted in two out of four hemocytometerquadrants, under 10× magnification. Only those cells which have nottaken up the trypan blue dye are counted. This process is repeated forthe second counting chamber. An average cell count per chamber is thusdetermined. Using means known in the art, the quadrant count values arechecked, logged, multiplied by 10⁴ to give cells/ml, and the totalamount of fluid (growth medium) necessary to suspend remaining cellaliquots is calculated accordingly.

After the desired concentration of cells in medium has been determined,additional cell aliquots from the monolayer are suspended in growthmedium via vortex and rocking and loaded into a Terasaki dispenser knownin the art. Aliquots of the prepared cell suspension are delivered intothe microtiter plates using Terasaki dispenser techniques known in theart. A plurality of plates may be prepared from a single cell suspensionas needed. Plates are then wrapped in sterile wet cotton gauze andincubated in an incubator box by means known in the art.

After the microtiter plates have been prepared, exposure of the cellstherein to active agent is conducted according to the followingexemplary protocol. During this portion of the inventive assay, theappropriate amount of specific active agent is tranferred into themicrotiter plates prepared as described above. A general protocol, whichmay be adapted, follows. Each microtiter plate is unwrapped from its wetcotton gauze sponge and microscopically examined for cell adhesion.Control solution is dispensed into delineated rows of wells within thegrid in the microtiter plate, and appropriate aliquots of active agentto be tested are added to the remaining wells in the remaining rows.Ordinarily, sequentially increasing concentrations of the active agentbeing tested are administered into progressively higher numbered rows inthe plate. The plates are then rewrapped in their gauze and incubated inan incubator box at 37° C. under 5% CO₂. After a predefined exposuretime, the plates are unwrapped, blotted with sterile gauze to remove theagent, washed with Hank's Balance Salt Solution, flooded with growthmedium, and replaced in the incubator in an incubator box for apredefined time period, after which the plates may be fixed and stainedfor evaluation.

Fixing and staining may be conducted according to a number of suitableprocedures; the following is representative. After removal of the platesfrom the incubator box, culture medium is poured off and the plates areflooded with Hank's Balance Salt Solution. After repeated flooding (withagitation each time) the plates are then flooded with reagent gradeethanol for 2-5 minutes. The ethanol is then poured off. Staining isaccomplished with approximately 5 ml of Giemsa Stain per plate, althoughvolume is not critical and flooding is the goal. Giemsa stain should beleft in place 5 min. ±30 seconds as timing influences stainingintensity. The Giemsa stain is then poured off and the plates are dipped3 times cold tap water in a beaker. The plates are then inverted, shakenvigorously, and air dried overnight (with plate lids off) on a rack on alaboratory bench. Cells per well are then counted manually or byautomated and/or computerized means, to derive data regardingchemosensitivity of cells at various concentrations of exposure. Oneparticularly useful computer operating environment for counting cells isthe commercially available OPTIMATE compiler, which is designed topermit an optical counting function well suited to computerized cellcounting procedures and subsequent calculations.

The above procedures do not change appreciably when cell growthpromoters are assayed rather than cell arresting agents such aschemotherapeutic agents. The present assay allows cell death or cellgrowth to be monitored with equal ease. In any case, optimization of useof the present system will involve the comparative testing of a varietyof candidate active agents, for selection of the best candidate forpatient treatment based upon the in vitro results. One particularlyadvantageous embodiment of the above described invention comprises atwo-stage assay for cytotoxicity followed by evaluation of longer-terminhibitory effect. Chemotherapeutic agents may thus be evaluatedseparately for both their direct chemotherapeutic effect as well as fortheir longer duration efficacy.

Identification of one or more active agents or chemotherapeutic agentsis peripheral to the present invention, which is intended for theefficacy screening of any or all of them as to a given patient.Literally any active agent may be screened according to the presentinvention; listing exemplary active agents is thus omitted here.

The essence of the invention thus includes the important feature of thesimplicity of the present system—cohesive multicellular particulates ofthe patient tissue to be tested are used to form cell monolayers; growthof those monolayers is monitored for accurate prediction of correlatinggrowth of the same cells in vivo; and differing concentrations of anumber of active agents may be tested for the purpose of determining notonly the most appropriate agent but the most appropriate concentrationof that agent for actual patient exposure (according to the calculatedcell growth rates). It is also important to note, in the context of theinvention, that the present system allows in vitro tests to be conductedin suspensions of tissue culture monolayers grown in nutrient mediumunder fast conditions (a matter of weeks), rather than with single cellprogeny produced by dilution cloning over long periods of time. In somecases, the present invention is a two stage assay for both cytotoxicityand the longer-term growth inhibitory.

Although the present invention has been described with respect tospecific materials and methods above, the invention is only to beconsidered limited insofar as is set forth in the accompanying claims.

1. A method for selecting an effective therapeutic agent for a cancerpatient comprising the steps of: (a) separating a tissue specimen intocohesive multicellular particulates, the tissue specimen being from saidcancer patient, wherein said cohesive multicellular particulates are notenzymatically dissociated cell suspensions or preparations; (b) growinga tissue culture monolayer from said cohesive multicellularparticulates; (c) treating a plurality of segregated sites, eachcontaining cells from said tissue culture monolayer, with a plurality ofactive agents at varied concentrations; and (d) measuring a response ofthe cells in the segregated sites to the plurality of active agents atvaried concentrations, to thereby identify an effective therapeuticagent for said patient.
 2. The method of claim 1, wherein said tissuespecimen is from a biopsy.
 3. The method of claim 1, wherein said tissuespecimen is from a surgery.
 4. The method of claim 1, wherein saidtissue specimen is malignant tissue.
 5. The method of claim 1, whereinsaid multicellular particulates have smooth cut edges.
 6. The method ofclaim 1, wherein said multicellular particulates are about 1 mm³.
 7. Themethod of claim 1, wherein said plurality of segregated sites is a platecontaining a plurality of wells therein.
 8. The method according toclaim 1 wherein the cells in step (c) are inoculated into the segregatedsites using a multiple well pipetting device.
 9. The method according toclaim 1 wherein step (c) comprises treating said plurality of sites witha plurality of active agents over a length of time adequate to permitdetermination of both initial cytotoxic effect and longer-terminhibitory effect of at least one of said plurality of active agents.10. The method according to claim 1, wherein said at least one activeagent is an anti-cancer agent, and wherein said response is sensitivityto said anti-cancer agent.
 11. The method of claim 8, wherein the cellsin step (c) are suspended in medium prior to inoculation.
 12. The methodof claim 1, wherein said plurality of active agents are chemotherapeuticagents.
 13. The method of claim 1, wherein said plurality of activeagents are selected from the group consisting of a radiation therapyagent, a radiation therapy sensitizing agent and a radiationameliorating agent.
 14. The method of claim 1, where said plurality ofactive agents are immunotherapeutic agents.
 15. The method of claim 1,wherein said plurality of active agents are for a non-malignantsyndrome.
 16. The method of claim 1, wherein said cells are fixed andstained prior to measuring the response of the cells.
 17. The method ofclaim 1, wherein measuring the response of the cells comprises a manualor automated counting of cells.
 18. The method of claim 17, wherein saidcells are counted using an optical counting function.
 19. The method ofclaim 17, wherein said cells are counted using a computer.
 20. Themethod of claim 1, wherein said response is cell growth or cell death.21. The method of claim 1, wherein said monolayer contains fewerfibroblasts as compared to a monolayer produced from suspended tumorcells grown in culture.
 22. A method for selecting an effectivetherapeutic agent for a cancer patient comprising the steps of; (a)separating a tissue specimen into cohesive multicellular particulates,the tissue specimen being from said cancer patient, wherein saidcohesive multicellular particulates are not enzymatically dissociatedcell suspensions or preparations; (b) growing said cohesivemulticellular particulates into a tissue culture monolayer, andsuspending cells of said tissue culture monolayer in medium; (c)directly aliquoting the suspended cells in a plurality of segregatedsites; (d) treating the cells in the segregated sites with a pluralityof active agents at varied concentrations, each treatment with an activeagent at a particular concentration being conducted in a segregatedsite; and (e) measuring a response of the cells from said monolayer toeach of the plurality of active agents at varied concentrations, tothereby identify an effective therapeutic agent for said patient. 23.The method of claim 22, wherein said tissue specimen is from a biopsy orsurgery.
 24. The method of claim 22, wherein said tissue specimen ismalignant tissue.
 25. The method of claim 22, wherein said multicellularparticulates have smooth cut edges.
 26. The method of claim 22, whereinsaid multicellular particulates are about 1 mm³.
 27. The method of claim22, wherein the segregated sites are a plate containing a plurality ofwells therein.
 28. The method of claim 22 wherein the suspended cellsare inoculated into the segregated sites using a multiple well pipettingdevice.
 29. The method of claim 22 wherein the cells of said monolayerare treated with the plurality of active agents over a length of timeadequate to permit determination of both initial cytotoxic effect andlonger-term inhibitory effect of at least one of said plurality ofactive agents.